Patterns of expression and translocation of the ubiquitin ligase SlrP in Salmonella enterica serovar Typhimurium

Abstract

SlrP is an E3 ubiquitin ligase that can be translocated into eukaryotic host cells by the two type III secretion systems that are expressed by Salmonella enterica serovar Typhimurium and are encoded in Salmonella pathogenicity islands 1 (SPI1) and 2 (SPI2). Expression of slrP and translocation of its product were examined using lac, 3×FLAG, and cyaA' translational fusions. Although slrP was expressed in different media, optimal expression was found under conditions that imitate the intravacuolar environment and promote synthesis of the SPI2-encoded type III secretion system. Translocation into mammalian cells took place through the SPI1- or the SPI2-encoded type III secretion system, depending on specific host cell type and timing. A search for genetic factors involved in controlling the expression of slrP unveiled LeuO, Lon, and the two-component system PhoQ/PhoP as novel regulators of slrP. Our experiments suggest that LeuO and Lon act through HilD under SPI1-inducing conditions, whereas PhoP directly interacts with the slrP promoter to activate transcription under SPI2 inducing conditions.

FIG 1

Expression of slrP in different culture media. β-Galactosidase activities were measured from overnight cultures of an S. enterica strain carrying a chromosomal slrP::lacZ translational fusion. (A) Bacteria were incubated overnight at 37°C in LB with 0.3 M NaCl without shaking (SPI1) or in LPM at pH 5.8 with shaking (SPI2). (B) Different concentrations of NaCl were used to test the role of osmolarity in sseK1 expression under SPI1-inducing conditions. (C) Activities were measured after growth in LPM with different pH and Mg²⁺ concentrations, as indicated. Means and standard deviations from three independent β-galactosidase measurements are shown.

FIG 2

Kinetics of translocation of SlrP into different cell types. Human epithelial HeLa cells (A), RAW264.7 murine macrophage-like cells (B), and NRK-49F normal rat kidney fibroblasts (C) were infected with derivatives of S. enterica serovar Typhimurium 14028 (wild-type [wt], ΔSPI1, ΔSPI2, and ΔSPI1 ΔSPI2 strains) carrying a chromosomal SlrP-CyaA′ fusion expressed under the native slrP promoter. To detect translocation, the level of cAMP was measured 1, 2, 4, 8, 16, and 24 h p.i. Bacteria were grown under SPI1-inducing conditions for most infections. Noninvasive bacteria were used specifically for infections of RAW264.7 cells for 4, 8, 16, and 24 h. Means and standard deviations from triplicate experiments are represented.

FIG 3

Regulation of slrP by the PhoQ/PhoP system. Expression of slrP was studied in different genetic backgrounds, as indicated, under SPI1 (A)- or SPI2 (B)-inducing conditions. Means and standard deviations from three independent β-galactosidase measurements are shown for strains carrying a slrP::lacZ fusion (top). Expression at the protein level was assessed by immunoblotting using strains expressing SlrP-3×FLAG (bottom). A monoclonal anti-FLAG antibody was used to detect the fusion protein, and a polyclonal anti-DnaK antibody was used as a loading control.

FIG 4

Regulation of slrP by lon, leuO, and hilD. (A) β-Galactosidase activity of slrP::lacZ in different genetic backgrounds: Δlon, T-POP leuO, and T-POP hilD. Tetracycline was added to the indicated cultures in order to express leuO or hilD from an internal T-POP promoter. (B) β-Galactosidase activity of slrP::lacZ in the presence and in the absence of LeuO or HilD. (C) Epistasis analysis of slrP expression. Effect of HilD expression on the β-galactosidase activity of slrP::lacZ in the presence and in the absence of Lon and LeuO.

FIG 5

Regulation of slrP by lon, leuO, and hilD at the protein level. The level of SlrP was studied by immunoblotting using strains expressing a 3×FLAG fusion. Extracts from stationary-phase cultures of these strains were resolved by SDS-PAGE (12%), and a monoclonal anti-FLAG antibody was used for Western blotting (upper bands). Polyclonal anti-GroEL or anti-DnaK antibodies were used as a loading control (lower bands). Molecular mass markers are indicated on the left. c, wild-type background. (A) Effects of overexpression and deletion of hilD on slrP expression. (B) Effect of Lon on slrP expression in the presence and in the absence of HilD. (C) Effect of LeuO on slrP expression in the presence and in the absence of HilD.

FIG 6

Analysis of the promoter region of slrP. (A) The sequence surrounding the transcriptional start site (+1) is shown. The consensus sequences for σ⁷⁰-dependent transcription, putative PhoP-binding site, ribosomal-binding site, and start of the coding region are indicated. (B) Alignment of the promoter region of slrP and six PhoP-activated genes with a similar architecture. Putative −10 and PhoP-binding boxes are indicated.

FIG 7

PhoP binds to the promoter of slrP. (A) Two fragments of DNA containing the promoter region of slrP, −460/+21 and −139/+21, were inserted into plasmid pIC552 to generate lacZ transcriptional fusions. These plasmids and derivatives carrying the indicated point mutations were introduced into S. enterica serovar Typhimurium strain 14028 (wild type) or a phoP-null mutant, and β-galactosidase activities were measured in cultures grown to stationary phase in liquid LPM. Means and standard deviations from two independent β-galactosidase measurements are shown. (B) Purified His₆-PhoP was phosphorylated in vitro using acetyl phosphate. DNA fragments containing the promoter region of slrP, the same region with a point mutation (T-40C), and the promoter regions of slyB and phoN, as positive and negative controls, respectively, were PCR amplified using fluorochrome-labeled primers and incubated with the indicated concentrations of phosphorylated His₆-PhoP (His₆-PhoP-P). Slot blotting was used to quantify binding. Results from a representative experiment of three independent experiments are shown.